The present invention, in some embodiments thereof, relates to antibodies which inhibit activity of metalloproteins, such as metalloproteases, and to methods which utilize the antibodies for treating diseases such as metastatic cancer which are associated with abnormal activity of a metalloprotein.
Enzymes are important medicinal targets for conditions ranging from pathogenic infections to cancer, thus many drugs produce their pharmacologic effects by inhibiting enzymatic activity. To obtain high selectivity, small molecule synthetic inhibitors as well as function blocking macromolecules (e.g. antibodies) are often targeted towards protein surface sites. However, this approach has limited impact due to the emergence of drug resistance mutations occurring under pathological conditions e.g. cancer and chronic infections. Such genetic changes take place in the form of rapidly acquired mutations that lead to loss of inhibition by the drug, while maintaining the original function of the protein target. Thus, although extremely challenging, targeting both key catalytic residues/elements and the enzyme surface appears to be the ultimate goal in drug development to assure potency and selectivity in vivo.
Remarkably, enzymes' endogenous inhibitors utilize molecular recognition mechanisms targeting both the protein active site and its surface. Among these are the natural protein inhibitors of matrix metalloproteinases (MMPs) namely, the intrinsic autoinhibitory pro-domains and the tissue matrix metalloproteinase inhibitors (TIMPs) which play important roles in regulating physiological and pathological cellular processes. The molecular and evolutionary designs of MMPs pro-domains and TIMPs utilize a highly potent inhibitory archetype mechanism involving direct binding of the catalytic metal ion which resides in the catalytic cleft and protein surface elements. Imitating these endogenous inhibitory interactions by antibodies (Abs) specific to metal ion and surface epitopes is a desirable proposition for specifically controlling metalloenzyme activity in vivo. Blocking metal activity of target metalloproteinases is the hallmark of rationally designed small molecule inhibitors. Yet, the design of selective small molecule inhibitors for individual MMPs has been shown to be highly challenging owing to the high structural similarities of the enzyme active site among family members.
Neurodegenerative diseases, including stroke, multiple sclerosis (MS) and related diseases, impact all aspects of society, causing great suffering and death, as well as imposing an enormous financial burden. Stroke results from a transient or permanent reduction in cerebral blood flow, while the earliest known event in the pathogenesis of MS lesions consists of the transendothelial migration of lymphocytes into central nervous system (CNS) white matter, which causes inflammation and disruption of the blood-brain barrier (BBB). Remarkably, each of these processes is thought to be largely mediated by the enzymatic activity of matrix metalloproteases (MMPs).
MMPs were thought to function mainly as enzymes that degrade structural components of the extracellular matrix (ECM). However, recent studies suggest that, beyond their classical connective-tissue-remodeling functions, MMPs also precisely regulate the function of bioactive macromolecules by proteolytic processing. Therefore, the potential effects of MMPs on cellular function are multifarious.
It has been shown that human T-cell migration across the subendothelial basal membrane (BM) in MS and stroke is mediated by the secretion of gelatinases A and B (designated MMP-2 and MMP-9), the production of which is controlled by independent genes. Moreover, it was demonstrated that mitoxantrone hydrochloride, which decreases progression of disability and clinical exacerbations in patients with MS, reduced matrix MMP-9 activity, as shown by zymography, polymerase chain reaction, and inhibitory studies. Rosenberg and co-workers demonstrated that selective inhibition of gelatinases by small molecule inhibitor SB-3CT reduced blood BBB disruption and prevent neuronal cell death. However, this compound suffers from low solubility in physiological solutions [Brain Res., 2007].
The subendothelial basal lamina is a unique structure that is composed predominantly of type IV collagen and laminin. Type IV collagen forms a nonhelical multilayer network that is resistant to nonspecific proteolytic degradation, but sensitive to gelatinase-mediated proteolysis. In vivo, gelatinases have been found to open the BBB, and pharmacological blockade of the active site of gelatinases was effective in inhibiting nervous system inflammation in MS animal models such as experimental allergic encephalomyelitis (EAE). In addition to the key role of gelatinase as a mediator of T-cell migration, other enzymatic activities of these proteases might also theoretically contribute to the disease process of MS. For example, gelatinase-mediated cleavage of myelin basic protein could contribute to accelerated antigen processing of this highly encephalitogenic protein. Furthermore, up-regulation of MMPs (especially gelatinase B) shortly after an ischemic stroke seems to contribute to subsequent brain damage by mediating degradation of the neurovascular matrix. Such proteolytic events may result in brain hemorrhage and neuronal apoptosis. Importantly, it was demonstrated, that INF-β suppresses gelatinase secretion and in vivo migration of human T-cells. Thus, controlling gelatinase activity in MS may contribute to treatment efficacy.
International Patent Application WO2004/087042 and WO2008/102359 teaches the generation of antibodies targeted at the catalytic zinc ion and the enzyme surface of MMPs.